A wide variety of scientific tests and procedures involve the analysis of photochemical reactions generally and the analysis of fluorescence in particular. Fluorescence is analyzed in research settings to identify and study both organic and inorganic compounds. Fluorescence is analyzed in clinical settings to obtain measurements in connection with immunoserology, microbiology, toxicology, clinical chemistry, histopathology, and coagulation assessment. In the industrial and agricultural arenas, fluorescence analysis is used during toxicity assays and contamination studies. Fluorescence is also analyzed in many contexts to study enzymes, amino acids, carcinogens, and a wide variety of other chemical compounds.
Fluorescence analysis also finds useful application in drug screening tests. The development of safe and effective drugs typically involves an extremely large number of tests. Fortunately, some of the required tests may be partially automated. For instance, tests to screen out ineffective compositions are often performed in large part by automatic machinery. Automation increases the number of compositions that can be tested, and also reduces the risk of error that often comes with repetitive tasks.
Automated screening tests typically begin by placing cell samples in several sample wells. The sample wells are small, cylindrical receptacles formed in a rectangular array in a transparent plastic sample plate. The typical sample plate contains an eight by twelve array of wells. Although the terms "row" and "column" are often used to designate a line of wells in the array, usage of these terms varies, with some sources referring to eight rows and twelve columns and others referring to eight columns and twelve rows. Without limiting the number of wells in a row, the latter usage is adopted herein. Thus, a typical row contains eight wells, but a row may also contain some other number of wells.
The cell samples are placed in the wells one well at a time or one row at a time by an automated liquid handler. A typical automated liquid handler includes a controller, at least one positionable row of pipettes, a subsystem for positioning the pipettes, and a subsystem for selectively directing a measured quantity of fluid through the pipettes. Some liquid handlers also include a subsystem for positioning a sample plate. The controller typically includes a programmed computer.
The pipettes are spaced apart from one another by the center-to-center distance between the sample wells in a standard sample plate. Thus, each pipette will fit in a separate sample well if the row of pipettes is placed over the row of wells and then lowered. At a minimum, the pipettes may be raised and lowered; many liquid handlers also permit the pipettes to be positioned in other ways.
In operation, cell samples to be studied are placed in the wells. The cell samples contain cells or cell contents which the unknown drug or other reagent being tested will act upon. The pipettes are positioned within a row of wells, and a first composition is injected into the cell samples through the pipettes. The composition, which is selected according to the test protocol, may include drugs or agonists. The pipettes are then positioned within the next row, and the process is repeated until the plate is filled.
Base line measurements are taken to calibrate the system. Thus, in the case of reactions which emit light, the level of light coming from each well is measured to determine the base line. All light emitted from the sample well may be monitored, or the light may be filtered before it is measured. For example, some drug screening tests involve the measurement of fluorescent light having a predetermined wavelength.
If the test protocol so requires, additional compositions may be added. The pipettes are rinsed, and then the second composition is added to the cell samples and the first composition so that additional reactions may be measured. Addition of the second composition is accomplished by positioning the pipettes in the wells that already contain the first composition and directing the second composition through the pipettes. The light coming from each well is again measured to gain experimental data. After rinsing, further compositions may be added and assessed in similar fashion.
In certain screening tests, the effectiveness of various compositions is measured by detecting and measuring the amount of light emitted from the sample well as the materials and cells in the sample wells react. In other screening tests, the amount of light transmitted through the well from a source external to the well is a useful measure of the desirability of particular compositions. Thus, instruments are needed which detect and measure at least a portion of the light from each sample well.
A conventional approach to detecting and measuring light involves directing a portion of the light emitted from each well in a row to a corresponding light detector in a row of light detectors. For instance, one system uses a row of fiber optic cables to carry light from a row of wells to a row of photomultiplier tubes. Each cable carries light from one well to one photomultiplier tube.
Such systems permit the light from an entire row of wells to be detected and measured at the same time, because the light from each well is directed to a different light detector. Processing an entire row at one time increases the number of wells that can be processed, which in turn reduces the time required to perform fluorescence analysis.
A problem in the art is that photomultipliers and other light detectors are typically expensive. Moreover, each light detector normally requires supporting equipment in order to detect and measure light. The necessary supporting equipment, which typically includes a power supply and a set of control electronics equipment, is often expensive and bulky. Thus, the use of a full row of light detectors adds substantially to the cost, size, and complexity of the light detection systems used in fluorescence analysis.
Another conventional system carries light from an entire plate of wells to a cooled charge-coupled device (CCD) camera. A CCD camera includes a rectangular array of picture elements known as pixels. The output of the entire sample plate is imaged on the CCD. Particular pixels corresponding to each well are then sampled to obtain light intensity readings for each well. Multiple wells in a row, or multiple rows of wells, can thus be processed in parallel and the time required for testing can be reduced.
Cooled CCD cameras are also extremely expensive in comparison to other types of light detectors. In addition, strong light sources, such as 1000 watt lights, are needed to illuminate the entire plate at once. Use of such strong lights is expensive, both because the lights themselves are costly, and because special optical elements must be used in connection with the strong light source. Moreover, costly secondary image processing is typically used to sample the pixels and to extract appropriate data from the CCD output.
Calcium is probably the most important and ubiquitous messenger linking plasma membrane depolarization to activation of intracellular biochemical events such as transmitter secretion or enzyme activation. Intracellular calcium ion (Ca.sup.+2) also plays a key role in mediating the actions of many transmitters, hormones, and drugs that act on plasma membrane receptors. At the heart of both voltage and receptor triggered calcium ion signalling is the intracellular or cytosolic free calcium ion concentration.
In order to measure calcium ion flow and change, molecular probes have been synthesized which can be placed within the cells to detect and reversibly bind messenger molecules and act as imaging agents. Fluorescence probes are the most popular because fluorescence is usually highly specific, extremely sensitive, and amenable to microscope detection.
Many clinically valuable drugs act by modulating calcium ion or calcium regulated enzymes. Channel blockers such as verapamil and nifedipine are used to treat angina and inhibit the entry of calcium into the heart or arterial smooth muscle cells by blocking calcium conducting channels.
One example of the importance of calcium ion flow is illustrated by the function of the regulatory parathyroid hormone (PTH). As mentioned above, intracellular calcium ion plays a pivotal role in regulating vaious cellular responses. The same is true with respect to extracellular calcium function, which is known to control various life-sustaining processes such as blood clotting, nerve and muscle excitability, and proper bone formation. PTH is known to act on kidney and bone tissue to increase the level of Ca.sup.+2 in the blood. Elevated levels of plasma Ca.sup.+2 in turn act in a negative feedback capacity to depress secretion of PTH.
The discovery of the Ca.sup.+2 receptor indicates the manner in which Ca.sup.+2 acts not only intracellularly to regulate various cellular functions but also extracellularly to regulate the activity of certain cells in the body. In both cases, Ca.sup.+2 interacts with a receptor protein. Those receptors within the cell are high-affinity Ca.sup.+2 binding proteins and are essentially ubiquitous. Those receptors on the cell surface are low affinity Ca.sup.+2 binding proteins and are restricted in their expression to certain specialized cells, many of which are intimately involved in bodily Ca.sup.+2 homeostasis.
Thus, it would be an advancement in the art to provide a system for detecting light during fluorescence analysis without using a separate light detector for each sample well in a typical row of sample wells. It would also be an advancement in the art to provide such a system which effectively detects fluorescent light in a predetermined range of wavelengths. It would be a further advancement to provide such a system which can be used effectively in combination with an automated liquid handler.
It would also be an advancement to provide an efficient and effective screening technique for determining the effect that various drugs or other reagents have on calcium function. It would be a related advancement to provide effective screening methods to determine whether a particular drug or reagent was a calcimimetic or calcilytic material, as those terms are defined herein.
Such a system is disclosed and claimed herein.